1. Field of the Invention
The present invention relates generally to a process for cloning genes into a suitable host for expression of enzymes. More particularly, this invention relates to a process permitting the expression of active, recombinant Acidothermus cellulolyticus cellulase enzymes from Escherichia coli.
2. Description of the Prior Art
Although an abundant biopolymer, cellulose is unique in that it is highly crystalline, insoluble in water, and highly resistant to depolymerization. Cellulose is also an important resource that can be used to produce ethanol for use as an alternative fuel; however, before cellulose can be utilized in fermentative processes to produce ethanol, it must be hydrolyzed to glucose (possibly cellobiose, depending upon the yeast strain chosen for fermentation). One method to produce glucose from cellulose containing biomass is to use cellulase enzymes. Cellulase enzymes have been isolated from a variety of bacteria and fungi. However, most of the research on cellulase enzymes has focused on the fungal enzyme systems, and in particular, the cellulases derived from mutants of the fungal strain Trichoderma reesei. This is because, under certain conditions, fungal cells can export relatively large amounts of cellulase enzymes into the culture broths (Eveleigh. 1987. Phil. Trans. R. Soc. Lond. 321(A): 435).
Several models for T. reesei cellulase action have been proposed, and although differences still exist among these models, it is clear that several enzymes act synergistically in the process of hydrolyzing cellulose completely to glucose. Two types of activity, the endo-1,4-.beta.-glucanases (EC 3.2.1.4) and the cellobiohydrolases (EC 3.2.1.91) are required to hydrolyze insoluble cellulose to produce the soluble dimer of glucose, cellobiose. A third activity, .beta.-glucosidase (EC 3.2.1.21) mediates the hydrolysis of cellobiose to glucose.
The production of ethanol from cellulose using T. reesei cellulase is costly. The fungi grow relatively slowly and sugar must be sacrificed both for cell growth and for the induction of enzymes (Bernard and Helinski. 1979. Methods Enzymol. 68:482). In addition, cellulase from T. reesei is significantly inhibited by cellobiose, and to a lesser degree, by glucose. This end-product inhibition can be ameliorated by simultaneous saccharification and fermentation (SSF) (Shoemaker et al. 1981Trends in the Biology of Fermentations for Fuels and Chemicals, Plenum Press: New York, N.Y., pp. 89). Compatibility with SSF technology requires new cellulases to be maximally active at mildly acidic pH (i.e., pH 4 to 5).
Because the cost of producing fungal cellulase enzymes is high, alternatives to fungal production of cellulases are needed for this process to become economically feasible. The activities of some fungal and bacterial cellulases have been investigated (Beguin and Gilkes, 1987. CRC Critical Rev. Biotechnol. 16:129). None of the bacteria studied to date is able to export cellulases to levels as high as those produced by today's improved strains of T. reesei, however. Genetically engineered Escherichia coli have been developed which dramatically increase the amount of recombinant enzymes (in general) produced in a given period of time (Bernard et al. 1979. Gene. 5:59). These E. coli strains grow much faster than fungi, with minimal medium costs. In addition, sugar is not required for induction of the enzymes, other signals (i.e., heat shock or chemical inducers) may be used to control overproduction of the enzymes.
The production of enzymes from fungi is very slow in comparison with those from bacteria, and extremely slow in comparison with bacteria that have been genetically engineered to overproduce enzymes. For example, a mutant of the fungus that has been obtained for optimal cellulase production required a total of 4 to 5 days for the production of 5 IFPU/mL of enzyme (Shell et al. 1990. Appl. Biochem. Biotech. 25:287), whereas using genetically engineered E. coli the enzyme xylose isomerase was produced at optimal levels within less than 1 day (Lastick et al. 1986. Biotech. Lett. 8:1). The differences in the amounts of enzyme produced between the two procedures is equally impressive; the genetically engineered cells were able to produce xylose isomerase at a level that represented 20% of the total cellular protein due to the introduction of a temperature controlled overproduction system. Other overproduction systems are currently available that use chemical signals to initiate the overproduction of the desired enzyme.
Currently, the most efficient cellulases have been isolated from strains of the fungus, T. reesei. However, endoglucanases from Thermomospora fusca (Wilson. 1988. Methods Enzymol. 160:314-323), Cellulomonas fimi (Gilkes et al. 1984. J. Biol. Chem. 259:10455-10459), Clostridium thermocellum (Beguin et al. In Biochemistry and Genetics of Cellulose Degradation, Academic Press: London, UK, 1988, pp. 267-282), and other bacteria have been cloned in E. coli with some success. No reports of the application of recombinant technology using genes from A. cellulolyticus have been observed in the published literature or patents.
Highly thermostable cellulase enzymes are known to be secreted by the cellulolytic thermophile A. cellulolyticus gen. nov., sp. nov., a bacterium originally isolated from decaying wood in an acidic, thermal pool at Yellowstone National Park and deposited with the American Type Culture Collection (ATCC) under collection number 43068 (Mohagheghi et al. 1986. Int. J. System. Bacteriol. 36:435-443). A. cellulolyticus is a unique thermophile whose taxonomy differs from the examples of bacteria given above. The cellulase produced by this organism is known to contain several different cellulase enzymes with maximal activities at temperatures of 75.degree. C. to 83.degree. C. In addition, the activity of the cellulases from A. cellulolyticus is much less inhibited by cellobiose than that found with cellulases from T. reesei, an important feature for hydrolysis of cellulose in the absence of yeast or .beta.-glucosidase. In addition, the cellulases from A. cellulolyticus are active over a broad pH range centered about pH 5, the pH at which yeasts are capable of fermenting cellobiose and glucose to ethanol. A novel cellulase enzyme secreted by the newly discovered microorganism is described in detail in the U.S. Pat. No. 5,110,735. In all, three distinct cellulases, the high and low molecular weight endoglucanases, and the E1 endoglucanase, have been described in this patent.
Recombinant bacterial enzymes can be used to either augment or replace the costly fungal enzymes currently used for cellulose degradation. The genes coding for A. cellulolyticus cellulases cloned into E. coli, or another industrial host, could provide an abundant source of highly active enzymes. The art of cloning A. cellulolyticus genes in E. coli or any other host organism has not been previously taught. Furthermore, is has not been previously taught that these enzymes may be useful in high temperature pretreatment of the cellulosic material prior to fermentation to ethanol.